Many human diseases and conditions are caused by gene mutations. Substantial effort has been directed towards the creation of transgenic animal models of such diseases and conditions to facilitate the testing of approaches to treatment, as well as to gain a better understanding of disease pathology. Early transgenic animal technology focused on the mouse, while more recent efforts, which have been bolstered by the development of somatic cell nuclear transfer (SCNT), have included larger animals, including pigs, cows, and goats. This technology has resulted in the production of, for example, pigs in which the gene encoding α-1,3-galactosyltransferase has been knocked out, in efforts to generate organs that can be used in xenotransplantation (see, e.g., Lai et al., Science 295:1089-1092, 2002). Further, this technology has resulted in the production of CFTR−/− and CFTR-ΔF508/ΔF508 pigs (see, e.g., U.S. Pat. No. 7,989,675 and U.S. patent application Ser. No. 12/283,980); and LDLR+/− and LDLD −/− pigs (see, e.g., U.S. patent application Ser. No. 13/368,312). Additional applications of this technology include the production of large quantities of human proteins (e.g., therapeutic antibodies; see, e.g., Grosse-Hovest et al., Proc. Natl. Acad. Sci. U.S.A. 101(18): 6858-6863, 2004). Substantial benefits may be obtained by the use of somatic cell nuclear transfer technology in the production of large animal models of human disease.
One example of a condition caused in part by a genetic mutation is Duchenne muscular dystrophy (DMD). DMD is a progressive neuromuscular disease caused by mutations in the X-linked dystrophin gene (DMD), which encodes the protein dystrophin (Hoffman, E. P., et al.; Cell 1987, 51 (6), 919-28). DMD is the most common muscular dystrophy, affecting approximately 1 in 3500 male births. DMD patients experience progressive weakness and degeneration in skeletal muscle, including the diaphragm, cardiac muscle, and some smooth muscle (Wallace, G. Q., et al. Annu Rev Physiol 2009, 71, 37-57).
Symptoms of DMD usually appear in male children before age five, but may appear in infancy. Progressive proximal muscle weakness of the legs and pelvis from loss of muscle mass is observed first, which spreads to the arms, neck, and other areas. Early signs may include pseudohypertrophy, low endurance, and difficulties in standing. As the condition progresses, muscle tissue experiences wasting and eventually undergoes fibrosis. By age 10, braces are usually required to aid in walking, with most patients becoming wheelchair dependent by age 12. Later symptoms may include abnormal bone development that lead to skeletal deformities, including curvature of the spine. Due to progressive deterioration of muscle, loss of movement occurs eventually leading to paralysis. The average life expectancy for patients afflicted with DMD is around 25 years.
There is no current cure for DMD. Current treatments include steroids, physical therapy, orthopedic appliances, and respiratory support, however none are directed at the underlying mechanistic defect. See Bushby, K.; et al.; Lancet Neurol 2010, 9 (2), 177-89; Bushby, K, et al.; Lancet Neurol 2010, 9 (1), 77-93. While these interventions have improved the lives of patients, DMD remains a lethal disease.
New gene- and cell-based therapies for DMD along with systemic delivery systems are rapidly advancing, however moving these therapeutic approaches to the clinic has been hampered by the lack of appropriate model systems. The available rodent models for DMD are not well suited for these applications due to their failure to develop an appropriate phenotype, and the naturally occurring canine models are problematic because of a wide variability in phenotype, limited choice of mutations, significant cost, and social acceptance concerns. Therapeutic strategies that have shown promise in these models have yet to be successfully translated to patients. An animal model that accurately and consistently replicates the clinical phenotype of human DMD and shares similarities to humans in size, anatomy, physiology, and genetics would be a transformative resource in bridging the substantial gap between models currently used for early-stage drug discovery and human clinical trials. In one embodiment, the present invention provides a new model of DMD in large, non-human mammals, for example, porcine. A successful large, non-human animal model that accurately replicates the manifestations of human DMD, and shares similarities to humans in size, anatomy, physiology, and genetics (for example, a porcine model) would have broad applicability and be a transformative resource in the DMD research community, and also provide the ability to develop much-needed models of other muscular dystrophies.
Furthermore, there is great interest in advancing medical devices, interventional strategies, and non-invasive diagnostic methods beyond their current state, but these fields are also limited by the current model systems. Rodent models are not well suited for most of these applications due to their size. Therefore, in one aspect of the invention, the transgenic animal model is a new model for DMD in a miniature pig breed. In one embodiment, the present invention accomplishes this in two steps by combining gene targeting and SCNT.